Results

To elucidate the role of Lbx in vertebrate myogenesis, we examined Lbx function in zebrafish. Zebrafish lbx2 transcripts appear in newly formed paraxial mesoderm and become restricted to adaxial cells, precursors of slow muscle. Slow muscles lose lbx2 expression as they differentiate, while a subset of differentiating fast muscle cells transiently expresses lbx2. Fin and hyoid muscle express lbx2 later. In contrast, lbx1b expression first appears lateral to the somites at late segmentation stages and is later restricted to fin muscle. Morpholino knockdown of Lbx1b and Lbx2 suppresses hypaxial muscle development. Moreover, knockdown of Lbx2 results in malformation of muscle fibers and reduced fusion of fast precursors, although no obvious effects on induction or specification are observed. Expression of myofilament genes, including actin and myosin, requires the engrailed repressor domain of Lbx2.

Conclusion

Our results elucidate a new function of Lbx2 as a regulator of myofibril formation.

Keywords

Background

Skeletal muscle progenitors are specified as a population of multipotent mesodermal cells. These cells subsequently commit to a muscle fate and differentiate into muscle fibers by cell fusion and assembly of contractile myofibrils composed of myosin thick filaments and actin thin filaments. Although the molecular mechanisms that regulate induction of muscle progenitors, commitment of these cells to the muscle lineage, and myoblast fusion are well characterized [1–5], relatively less is known about the mechanisms that regulate the formation of myofibrils [6, 7].

The ladybird (Lbx) protein is a member of the homeobox transcription factor family, characterized by an N-terminal engrailed repressor domain. In Drosophila, lbx is expressed by progenitor cells, founder cells, and syncytial precursors of a larval somatic muscle, the segmental border muscle (SBM, muscle 8) [8, 9]. Embryos lacking lbx function have missing muscle fibers, unfused myoblasts, or abnormally shaped cells in the SBM. Ectopic lbx expression leads to the formation of enlarged or duplicated SBMs [8]. Recent studies showed that Lbx is also required for establishment of morphological, ultrastructural, and functional properties of leg muscles in Drosophila [10]. Thus, the lbx gene functions in fusion of myoblasts, development of muscle fibers, and the establishment of muscle morphology in Drosophila.

In mouse, two Lbx genes, Lbx1 and Lbx2, have also been identified [11]. Lbx1 is expressed in a subset of hypaxial myoblasts that migrate into the limbs, tongue, and diaphragm. Lbx2 is expressed in the central nervous system, in neural crest derived structures such as dorsal root ganglia and other parts of the peripheral nervous system, and in the urogenital system [11]. Loss of Lbx1 function results in lack of specific limb musculature, attributed to migration defects [12, 13]. Although, Lbx2 knock out mice develop relatively normally, one study suggested that Lbx2 functions in neural-derived tissues under the regulation of Pax3 [14]. Lbx1 has also been studied in Xenopus, where it represses myod expression and promotes myoblast proliferation before the onset of terminal differentiation [15]. Thus in both mammals and frogs, Lbx1 is thought to function in migrating myoblasts, whereas functions of Lbx2 are still unclear [14].

Many aspects of the molecular pathway that regulates myogenesis are conserved in Drosophila and vertebrates [5, 16]. However, because studies of vertebrate Lbx have focused mainly on migration or proliferation of myoblasts in hypaxial myogenesis, little is known about whether Lbx functions in other aspects of muscle development that have been characterized in Drosophila, such as fusion of myoblasts, formation of fibers, or the establishment of muscle shapes. To study these potential roles of Lbx in vertebrates, we examined lbx gene function in zebrafish. Zebrafish axial skeletal muscles contain four fiber types; slow muscle, muscle pioneers, fast muscle, and medial fast fibers. Slow muscle cells are located superficially just under the skin, with fast muscle cells located deeper [1]. Muscle pioneers are located near the horizontal myoseptum that separates dorsal and ventral parts of the myotomes, and medial fast fibers are also located medially in the somite. We previously showed that adaxial cells located adjacent to the notochord are precursors of slow muscle cells and muscle pioneers [17, 18]. Adaxial cells are specified to form slow or muscle pioneers by Hh signaling from the notochord [19], Then, slow muscle precursors migrate to the lateral surface of the somite [18]. After the somite forms, non-adaxial muscle precursors in the segmental plate differentiate into fast muscle cells. During this period, both slow and fast muscle precursors are dynamically rearranged in the somite [20, 21].

The zebrafish genome contains three lbx genes [22].lbx2, previously named lbx1, is expressed in the ventral region of the somite, in the hindbrain, and in the fin bud [23]. lbx1a is expressed in the nervous system and fin bud [24]. Analysis of lbx1b expression in zebrafish has not been previously reported[22].

Here, we show that zebrafish lbx2 expression first appears at late gastrula stages (70%–80% epiboly) in cells near the margin that later form head muscle and pronephros. Subsequently, lbx2 expression is present in the paraxial mesoderm and adaxial cells, which are the precursors of slow muscle and muscle pioneers [1, 18]. Although a subset of adaxial cells migrates into the somite during segmentation stages[18], lbx2 is not detected in slow muscle cells during or after their migration. As development proceeds to late segmentation stages, a subset of fast muscle cells in both dorsal and ventral regions of the somite turns on lbx2 expression that subsequently disappears by the second day of development. Thus, myoblasts in both the slow and fast muscle lineages express lbx2 transiently. As development proceeds to the end of the second day, lbx2 expression is detected in fin bud and hyoid, similar to Xenopus lbx1 expression. In contrast to lbx2, lbx1b expression is not detected during gastrulation, but first appears in the hindbrain and caudal regions of the neural tube around the 5-somite stage. As development proceeds, a subset of cells lateral to the somites (presumptive fin bud) begins to express lbx1b, whereas somite cells never express lbx1b. Later, lbx1b expression is present in the fin buds. Thus, myoblasts only in fin lineages express lbx1b. Morpholino analysis shows that both Lbx1b and Lbx2 function in hypaxial myogenesis. In addition, although Lbx2 is not required for induction of myogenesis or specification of zebrafish skeletal muscle cell fates, Lbx2 is required for normal fusion of fast muscle precursors, as in Drosophila. Lbx2 also plays an important role in the formation of myofibrils by regulating expression of filament genes, such as myosin and actin. We further demonstrate that the engrailed repressor domain of the Lbx2 protein is required for this induction of filament genes. Thus, although previous studies in vertebrates have suggested that Lbx1 functions in proliferation and migration of muscle precursors, our results demonstrate that vertebrate Lbx2 also plays an important role as a regulator of muscle cell differentiation.

Results

Slow and fast muscle precursors transiently express lbx2mRNA

To study the function of lbx genes in vertebrate skeletal muscle development, we examined expression of lbx1b and lbx2 in zebrafish. Previous analysis of lbx1a [24] showed that it is not expressed in the somites, so we excluded it from this study. lbx2 expression becomes detectable by the 70%–80% epiboly stage (Fig. 1A). Double labeling with no tail (ntl), which marks the blastoderm margin, reveals that lbx2 first appears adjacent to the margin (Fig. 1H) in the region that later contributes to head muscle and pronephros [25]. By the end of bud stage, lbx2 expression appears in paraxial mesoderm and adaxial cells (Fig. 1C, J, brackets), precursors of slow muscle cells and muscle pioneers [18]. lbx2 expression is not detected in the somites (Fig. 1D, K, rostral region), even though a subset of adaxial cells, the slow muscle precursors, migrates through the somites during segmentation stages [18, 20, 21].

Unlike lbx2, lbx1b expression is not detected during gastrulation. lbx1b expression first appears within a dorsal intermediate region of the neural tube by the 5-somite stage (Fig. 1N, arrow), and this expression extends along the rostral caudal axis by late segmentation stages (Fig. 1O, arrow). As development proceeds, a group of cells lateral to the somites (presumptive fin bud) begins to express lbx1b (Fig, 1P, Q, arrowhead), whereas muscle precursors in the somites do not express lbx1b. Subsequently, lbx1b expression is detected in the fin bud (Fig. 1R, arrowheads). Unlike lbx2, the hyoid does not express lbx1b (Fig. 1R). Thus, during myogenesis, lbx2 expression appears in paraxial mesoderm, precursors of slow and fast muscles of the somite, fin bud, and hyoid, whereas lbx1b is detected only in myoblast lineages of the fin.

Conserved function of lbxin hypaxial muscle development

To examine Lbx function in myogenesis, we knocked down Lbx2 or Lbx1b activity using specific splice blocking morpholino oligonucleotides (MOs). To confirm the specificity of the MOs, we performed reverse transcriptase PCR on mRNA from MO injected embryos. We find that injection of lbx2 or lbx1b splice donor MO results in production of aberrantly spliced lbx2 or lbx1b transcripts, respectively (Fig. 2A), indicating that splicing of lbx2 and lbx1b is blocked by the specific lbx splice donor MOs.

Previous studies suggested that Lbx1 regulates the migration of limb, hypoglossal, and head muscle precursors in mouse and Xenopus [12, 13, 15]. Our observation that zebrafish lbx2 appears in fin bud and hyoid muscles suggests that Lbx2 may be involved in regulation of hypaxial and head muscle development. We find that inactivation of Lbx2 blocks formation of the fin bud and blocks myod expression in hyoid muscles (Fig. 2.B–E, arrows and arrowheads respectively). Embryos injected with lbx2-MO express fgf8 normally in the ectoderm (Fig. 2H–K, arrows)[26], indicating that other aspects of fin bud development are unaffected by MO injection. In addition to lbx2, zebrafish lbx1b is also expressed in the fin bud (Fig. 1P–R). Injection of lbx1b-MO results in decreased myod expression in the fin bud, and again, fgf8 expression is normal (Fig. 2F, G, L, M). Thus, zebrafish Lbx2 and Lbx1b are both required for proper development of hypaxial muscles that are derived from lbx2 and/or lbx1b expressing myoblasts, similar to Xenopus and mouse Lbx1 [12, 13, 15].

Functional roles of Lbx2 in the induction of myogenesis and the migration of slow muscle precursors

Our observation of transient expression of lbx2 in paraxial mesoderm and adaxial cells at the end of bud stage suggests that Lbx2 might be involved in specification of muscle cells at this stage. [27]. However, we find no obvious changes in expression of myogenic regulatory factors such as myod expression in adaxial cells or myf5 expression in paraxial mesoderm in lbx2 knock down embryos (Fig. 3A–D), although a few myod expressing cells fail to incorporate properly into the adaxial cell layer (Fig. 3H, arrow) and the distinctive cuboidal shape of the adaxial cell pseudo-epithelium is somewhat disrupted in (Fig. 3E, F, arrow). Transplantation analysis showed that, although morphogenesis of cells lacking Lbx2 activity is affected, they still express myod when located adjacent to the notochord (see Additional file 1). These results indicate that Lbx2 is not involved in the induction of myogenesis.

During segmentation stages, the slow muscle precursors, a subset of adaxial cells, migrate radially away from the notochord to form the superficial layer of slow muscle [18]. Because adaxial cells transiently express lbx2 (Fig. 1J, K), we examined whether Lbx2 is involved in their migration. Cross sections of lbx2-MO injected embryos indicate that muscle precursors migrate properly through the somite to form the superficial layer (Fig. 4D, H, L). Transplantation analysis also showed that cells lacking Lbx2 function migrate properly to the superficial layer in uninjected embryos (not shown). Thus, in the absence of Lbx2 activity, mesodermal cells form muscle precursors, as indicated by myod and myf5 expression, and slow muscle precursors migrate normally to the lateral surface of the somite.

Discussion

Previous studies suggested that Lbx1 regulates the migration of limb and hypoglossal myoblasts in mouse [12, 13]. Recently, an additional function of Lbx1 was reported, whereby Lbx1 represses myod expression and promotes myoblast proliferation before the onset of terminal differentiation [15]. In contrast, functions of Lbx2 are poorly understood, because Lbx2 deficiency does not impair mouse development [14]. Here, we demonstrate in zebrafish that lbx2 is expressed in mesodermal cells, migratory slow muscle precursors, fin bud, and hyoid [23], whereas lbx1b is expressed only in fin bud during myogenesis. Loss of Lbx2 function does not apparently affect induction of myogenesis, slow muscle migration, or proliferation, but rather reduces cell fusion, as in Drosophila, and, furthermore, Lbx2 knockdown blocks differentiation of myofibrils due to reduced expression of specific thin and thick filament genes.

Slow and fast muscle precursor cells transiently express Lbx2

lbx2 expression appears by the end of bud stage in paraxial mesoderm and in adaxial cells, the precursors of slow muscle including muscle pioneers. A subset of adaxial cells migrates through the somite during segmentation stages [18], but lbx2 is not expressed in nor required for migration of these cells. Interestingly, a subset of fast muscle cells begins to express lbx2 in both epaxial and hypaxial domains by the end of segmentation stages. A previous study in zebrafish concluded that lbx2 expression is restricted to ventral regions of the somites [23]. This discrepancy could be due to the rather weak expression of lbx2 in the epaxial domain (Fig. 1E), which may have been missed in the earlier study. By 48 hpf, expression of lbx2 disappears from somites in the trunk. Similarly in Xenopus, the ventrolateral region of rostral trunk somites, which contains precursors of hypaxial body wall, expresses lbx1, and this expression later becomes weak and eventually lost [15]. Satellite cells in mouse that express Lbx1 also extinguish expression during myogenic differentiation [34]. Thus, Lbx gene expression may be transient during myogenesis in all species.

During muscle development in chick, the myotome forms in three waves [35, 36]. The first wave of early post-mitotic progenitors appears along the entire dorsomedial aspect of the epithelial somite. Then in the second wave, cells migrate from all four lips of the dermomyotome, although myofibers are generated from only the rostral and caudal edges. During the third wave, there is a balance between differentiation and proliferation. Similarly, zebrafish muscle development can be divided into several steps. First, epithelial adaxial cells, precursors of slow and muscle pioneers, are specified to be slow or muscle pioneers by Hh signaling from the notochord [19]. Then, slow muscle precursors migrate to the lateral surface of the somite [18]. Second, a wave of fast muscle morphogenesis is induced by the migration of the slow muscle precursors [37]. Finally, new slow-muscle fibers are added in growth zones near the dorsal and ventral extremes of the myotome, and this muscle growth continues into larval life [38]. We demonstrated that lbx2 expression first appears in adaxial cells. Adaxial cells lose lbx2 expression before they incorporate into the somite. Then, a subset of fast muscle precursors begins to express lbx2. Further analysis is required to determine whether Lbx2 is transiently expressed in all or only a subset of muscle precursors and whether such transient expression is linked to particular waves of muscle development.

Migration of slow muscle precursors in Lbx2 knockdown embryos

In mouse, disruption of Lbx1 causes a severe loss of limb muscle, because muscle progenitors fail to migrate [13]. We showed that disruption of Lbx2 activity causes decreased myod expression in fin bud and hyoid muscles (Fig. 3), suggesting that Lbx2 could be involved in migration of these precursors in zebrafish. Additionally, we previously reported that a subset of adaxial cells migrate laterally to form slow muscle [18]. The expression of lbx2 in adaxial cells raised the possibility that Lbx2 may regulate migration of slow muscle precursors, too. Although transplantation analysis showed that the early movement of cells lacking Lbx2 is disrupted during gastrulation (see Additional file 1), slow muscle precursors eventually migrate radially (Fig. 4). Therefore, although it is likely that Lbx2 contributes to the migration of fin and head muscle precursors, Lbx2 is not required for migration of slow muscle precursors.

A molecular link between Lbx2, a transcriptional repressor, and filament gene expression

The Lbx1 protein contains the engrailed repressor domain [39] and Lbx1 protein that lacks the engrailed domain fails to suppress myod expression in Xenopus [15]. Moreover, Lbx1 interacts with Corl1, a transcriptional corepressor [40]. These observations strongly suggest that Lbx1 functions as a transcriptional repressor. We find that Lbx2 that lacks the engrailed domain fails to induce atca1 and mybpc1 in lbx2-MO injected embryos, and also fails to suppress mef2c and myogenin in lbx2-MO injected embryos (Fig. 7). This result indicates that the engrailed repressor domain is required for function of Lbx2 in the formation of myofibrils. However, our observation that lbx2-MO leads to a decrease in filament gene expression suggests that filament genes cannot be direct targets of Lbx2. Instead, this result indicates that Lbx2 acts through an intermediate factor (or factors) to regulate expression of filament genes.

Myogenin and Mef2 are known to function in filament formation [7, 41, 42], and we showed that expression of both myogenin and mef2c is reduced by overexpression of Lbx2. Thus, Myogenin and Mef2c may act downstream of Lbx2 to regulate myofilament gene expression during formation of myofibrils. However, Myogenin and Mef2c are thought to function as transcriptional activators. Our observations that myogenin and mef2c expression is upregulated in lbx2-MO injected embryos, whereas filament gene expression is decreased by lbx2-MO, suggest that there must be other factors downstream of Lbx2, most likely additional transcriptional repressors. Thus, further studies are required to identify the proximal downstream mediators of Lbx2 function.

Conclusion

Several steps are required for muscle progenitors to form functional skeletal muscle: (1) formation of a population of multipotent mesodermal cells, (2) specification and commitment to a muscle fate, and (3) differentiation accompanied by cell fusion and assembly of contractile myofibrils. The precise timing of these steps requires a fine balance between transcriptional activation and repression [43, 44]. Although many transcriptional activators and repressors involved in commitment, specification, and differentiation of muscle precursors have been identified [45], the transcriptional network that regulates terminal differentiation is still incompletely understood. Our study has identified Lbx2 as a regulator of myofibril formation through its action on expression of myofilament genes, including myosin and actin. A recent study showed that undifferentiated satellite cells express Lbx1, and Lbx1 expression diminishes as cells differentiate [34]. In addition, it is known that Lbx1 upregulates Pax7 expression and downregulates Myod in satellite cells [34], and we also find suppression of pax7 expression in lbx2-MO injected embryos (see Additional file 6). Thus, it is possible that Lbx1 maintains the immature state of satellite cells by regulating Pax7. Based on these observations, we suggest that the general function of Lbx2 may be to aid differentiation of myoblasts by promoting fusion and myofilament gene expression.

Methods

Animals

Embryos were obtained from the University of Oregon zebrafish facility, produced using standard procedures [46] and staged according to standard criteria [47]. The wild-type line used was AB.

Plasmid construction

Gene-specific primers to amplify full-length zebrafish lbx2 (AJ29516) and part of lbx1b cDNA were designed based on the available Ensemble zebrafish genome assembly (Zv4).

Microinjection

mRNA was dissolved in double distilled H2O to final concentrations of 30 ng/μl to 50 ng/μl. Phenol red was added to the solution. Approximately 1 nl of RNA or DNA was injected at the one-cell stage using published procedures [51]. lbx2-MO was directed to the translation start site, splice donor site (Gene Tools, LLC), lbx2-ATG MO: TCATGTCTTTAGAGCTGGAGGTCAT, lbx2-splice donor MO: TTATGAACTTTTTACCTTCTGCTGC, lbx1b-splice donor MO: ACACCGGGCCTTGTGTTTACCTTCT. Stock solutions were resuspended at 2.5 μg/μl.

To obtain mosaic expression, we performed the plasmid injected (100 ng/μl).

Antibody labeling and quantification of muscle fiber numbers

Labeling with F59, EB165, 4D9 and Prox1 was as previously described [3, 5, 17]. The primary antibodies were mAb F59 (anti-MyHC, slow muscle) at 1:20, mAb EB165 (anti-MyHC, fast muscle) at 1:5000, mAb 4D9 (anti-Engrailed) at a dilution of 1:20, rabbit anti-Prox1 (AngioBio Co.) at a dilution of 1:500. Secondary antibodies were AlexaFluor-594 goat anti mouse IgG at 1:1000 and AlexaFluore-488 goat anti-rabbit IgG at 1:1000 (Molecular Probes). The images ware collected using a LSM 5 PASCAL confocal microscope configured around an AXIO Imager M1 upright microscope. The numbers of slow muscle and muscle pioneers were calculated from the total number of cells labeled by the Prox1 antibody (slow muscle) and the total number of cells labeled by both the Prox1 and 4D9 antibodies (muscle pioneer cell) per embryo counted in four somites over the extended yolk at 24 hpf. Data represent the average number of slow muscle cells or muscle pioneer cells per somite. To quantify the phenotypes of muscle fibers, we examined somites over the extended yolk in 3–5 embryos. Fibers that were positioned in the middle of the dorsal half of the somite were measured. The lengths of muscle fibers were measured from the anterior somite border to the end of each fiber (Fig. 4B arrow). Because fiber thickness depends on how many sarcomeres line up in parallel [52], we measured the diameter of each fiber from its dorsal to its ventral edge (Fig. 4B arrowhead).

Copyright

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.